Prokaryotic expression and activity analysis of insulin-like growth factor-I from Epinephelus septemfasciatus

In order to study the underlying molecular mechanisms for growth of Epinephelus septemfasciatus,the insulin-like growth factorⅠ(IGF-Ⅰ)mature peptide sequence was amplified from E.septemfasciatus liver with RT-PCR method.It was predicted that the mature peptide was composed of 210 base pairs,which encodes 70 amino acid residues and consists of B-C-A-D four functional domains.The matured peptide fragment was subcloned into the expression vector pET-28a and was successfully expressed in E.coli BL21(DE3)cell.The result of SDS-PAGE analysis indicated that the fusion protein expressed in the form of inclusion bodies with molecular weight of 11 ku and maximally amounted to 51.8% of the whole protein in the E.coli cell 3 hours after being induced with IPTG.The purified recombinant protein was obtained through denaturation,purification and refolding.The Western-blotting indicated that the recombinant protein had specifically been recognized by 6×His antibody.The proliferation experiment showed that the purified IGF-Ⅰ fusion protein could significantly promote the proliferation of breast cancer cells MDA231,and this indicated that it has biological activity.